CAMBRIDGE, Mass.—Organoids, miniature, 3D versions of organs grown in
vitro from embryonic or induced pluripotent stem cells, are a promising resource for a variety of types of lab work, from watching disease progression
to testing toxicity on a small scale in miniaturized livers or kidneys. As technology—and techniques—have progressed, scientists have moved from
2D to 3D models, experimenting with culture components and bioscaffolds to allow cells to grow more organically. Given the complexity of the brain and the
variety of cells within the central nervous system, generating accurate brain organoid models has been more difficult, and only the last decade has shown
significant progress, with successful development of grey and white brain tissue in 3D models happening only as recently as 2014 (check out “Tufts sees a touchdown with 3D tissue” for more information).

And in advancing things even further, a joint team from Harvard
University and the Broad Institute of MIT and Harvard working with human brain
organoids has found that a longer culture time can offer more accurate and mature organoids, ones that present with multiple different cell types found in
human brains. Their work was published in the paper “Cell diversity and network dynamics in photosensitive human brain organoids,” which appeared
in Nature.

In general, most brain organoids are cultured over the course of days or weeks. The Harvard-
Broad team adjusted their culture conditions in order to produce organoids that would mature over a matter of months—up to nine months or longer. They
were also able to offer in their paper the largest molecular map to date of the different kinds of cells produced in brain organoids and their
reproducibility.

“In the past, researchers have used a cocktail of factors to develop pluripotent stem cells into different
types of cells from the nervous system—neurons, astrocytes, sometimes even specific classes of neurons,” said senior author Paola Arlotta, an
institute member in the Stanley Center for Psychiatric Research at the Broad Institute, co-director of the nervous system disease program at the Harvard Stem
Cell Institute, and professor of stem cell and regenerative biology at Harvard University. “But the brain contains an incredible diversity of cell
types that interact and form connections. We took on the challenge of investigating to what extent such complexity and diversity of cell types can be
produced in the dish, and how closely the organoid cell types mirror those in the endogenous tissue.”

As noted on
Arlotta's faculty page, her work is focused on studying the mammalian cerebral cortex, neuronal diversity in that system and how that diversity affects
the function of certain cells. She cites the lab's work with organoids as well, noting that they have “been building in-vitro models that
resemble the cellular complexity, tissue architecture and local connectivity of the developing human cerebral cortex, which can become a platform for
understanding higher-order circuit function and dysfunction that is affected in neurodevelopmental and neuropsychiatric cortical disease.”

Giorgia Quadrato, first author on the paper and a postdoctoral fellow in Arlotta's lab, and his fellow researchers
adjusted lab protocols in order to allow for organoids that would develop over a longer period of time from induced pluripotent stem cell lines. Single-cell
RNA sequencing (also known as “Drop-seq”) was utilized to analyze gene expression in more than 80,000 individual cells isolated from 31 organoids
in order to categorize the different cell types these mini organs create. Their results showed that the more time the organoids had to develop, the more cell
types they formed.

The cell types generated included subtypes of neurons and progenitors of the cerebral cortex,
as well as many cell types comprising the visual system—upon further analysis, the team found a nearly complete collection of cell types seen in the
human retina, including photoreceptor-like cells that caused a response when stimulated with light. Based on their findings, the researchers believe these
models “may offer a way to probe the functionality of human neuronal circuits using physiological sensory stimuli,” as noted in the paper's
abstract.

“The cellular diversity that the organoids generated stunned all of us,” said paper co-author Steve
McCarroll, an institute member at Broad, director of genetics for the Stanley Center for Psychiatric Research, and associate professor of genetics at Harvard
Medical School. “The ability of stem cells within organoids to generate so many of the brain’s cell types—using their own genetic and
molecular instruction book—evokes how development works inside the body.”

Roughly eight months into culturing,
the neurons in the organoids developed dendritic spines, which protrude from the dendrites where synapses form and are a feature of mature neurons; thus far,
it's been difficult to replicate them in culture.

According to McCarroll, “The presence of dendritic spines in these organoids
is an important step for studying development and disease. Genetic studies indicate that disorders such as schizophrenia involve dysfunction at synapses and
perhaps in the regulation or pruning of dendritic spines. An experimental model that develops spines can open the door to understanding how genes, and
perhaps new pharmacological therapies, shape synaptic biology.”

Moving forward, the team plans further investigate
options for maturing organoids more rapidly and guiding their anatomical organization as they mature, as well as hopefully reducing variability between
organoids to better study neuronal maturation and networks.

As for Arlotta's lab, her faculty page also notes
that “In the long term, our work aims at developing approaches to aid neuronal regeneration in neurodegenerative diseases of the cortical output
circuitry, and at understanding and modulating neuronal function in neuropsychiatric diseases affecting the cerebral cortex.”